NO (Nitric Oxide, NO) is widely distributed in living organisms, including the nervous, circulatory, respiratory, digestive, urogenital and other systems, especially more abundant in the nervous tissue. It plays a signaling role as an intercellular and intracellular information substance, and is a new type of biomessenger molecule, which plays an important role in the physiological and pathological processes of the organism.
Principle
NO is a kind of extremely unstable biological free radicals, in the body or in aqueous solution, it is very easy to oxidize and generate NO22- and NO33-, NO33- can be reduced to NO22- through the cadmium, NO22- under acidic conditions and diazonium salt sulfonate amine to generate diazo compounds to generate diazo compounds, and then with naphthalene-based vinyl diamine to carry on the coupling reaction, to generate colorful compounds, the absorbance value is determined by spectrophotometer at 550 nm, and the standard curve can be calculated from the standard curve. The absorbance value is measured at 550 nm by spectrophotometer, and a standard curve is made, through which the NO content can be calculated.
Operation method
Determination of Nitric Oxide using the kit
Principle
NO is a kind of extremely unstable biological free radicals, in the body or in aqueous solution, it is very easy to oxidize and generate NO22- and NO33-, NO33- can be reduced to NO22- through cadmium, NO22- under acidic conditions and diazonium salt sulfonate amine to generate diazo compounds to generate diazo compounds, and then with naphthalene-based vinyl diamine to carry on the coupling reaction, to generate colorful compounds, through spectrophotometer at 550 nm to determine the absorbance value and make a standard curve, through the standard curve can be calculated NO content. The absorbance value is measured at 550 nm by spectrophotometer, and a standard curve is made, through which the NO content can be calculated.
Materials and Instruments
Balance, Spectrophotometer/Enzyme Labeler, Cryogenic Centrifuge Move I. Sample Processing
Water bath/incubator, glass cuvettes/96-well plates
Pipette gun, mortar/homogenizer, ice and distilled water, EP tubes
Nitric Oxide (NO) Assay Kit (Soluble BC1475)
Preparation of solutions:
1. Reagent 1: Place the powder in a glass vial inside the reagent bottle. Add 2.5 mL of distilled water and mix well before use, and store at -20 ℃ for two weeks to avoid repeated freezing and thawing;
2, color development solution: before use in accordance with the required amount of experiments according to liquid A : liquid B = 1 : 1 fully mixed, ready to use;
3、 Standard: 10 μmol/mL sodium nitrite.
1, tissue samples: according to the mass (g): extract volume (mL) 1 : 5 ~ 10 ratio (recommended to weigh 0.2 g of sample, add 1.0 mL of extract) to add extract, ice bath homogenization, centrifugation at 4 ℃, 12,000 rpm, 15 min, discard the precipitate, take the supernatant on ice to be measured.
2, cell samples: according to the number of cells ( 104 ): the volume of extraction solution (mL) 500~1000:1 ratio (recommended 10 million cells to add 1.0 mL of extraction solution) to add the extraction solution, ice bath ultrasonic cell crushing (power 300 w, ultrasonication 3 s, interval 7 s, the total time of 3 min), and then in 4 ℃, 12000 rpm, centrifugation for 15 minutes, discard precipitate, take the supernatant on ice to be tested. Then centrifuge at 4 ℃, 12000 rpm for 15 min, discard the precipitate, and put the supernatant on ice to be measured.
3、Liquid sample: Measure directly, if the liquid is turbid, centrifuge the supernatant for measurement.
Measurement steps
1、Warm up the spectrophotometer/enzymometer for more than 30 min, adjust the wavelength to 550 nm, and adjust the zero with distilled water.
2、Place the reagent on ice.
3、Dilution of standard solution: dilute the standard with distilled water to 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 μmol/mL. the standard dilution can be referred to the following table:
| No. | Concentration before dilution (µmol/mL) | Volume of standard liquid (µL) | Volume of distilled water (µL) | Concentration after dilution (µmol/mL) |
| 1 | 10 | 100 | 900 | 1 |
| 2 | 1 | 200 | 800 | 0.2 |
| 3 | 0.2 | 500 | 500 | 0.1 |
| 4 | 0.1 | 500 | 500 | 0.05 |
| 5 | 0.05 | 500 | 500 | 0.025 |
| 6 | 0.025 | 500 | 500 | 0.0125 |
| 7 | 0.0125 | 500 | 500 | 0.00625 |
| 8 | 0.00625 | 500 | 500 | 0.003125 |
Note: 100 µL of standard solution is required for each standard tube in the experiment.
4. Operation table:
| Reagent name (µL) | Blank tube | Measurement tube | Standard tube |
| Distilled water | 120 | -Distilled water | 20 |
| Standard liquid | - - - - - - - - - - - - - - - - - - - - - - - | - | 100 |
| Sample | -Samples | 100 | - |
| Reagent I | Reagent I - | 20 - | -Vortex mixing |
Vortex and mix well, place in 37 ℃ constant temperature incubator/water bath for 60 min.
| Reagent II | 20 | 20 | 20 |
Vortex and mix well, let stand at room temperature for 5 min, centrifuge at 3500 rpm for 10 min, take the supernatant to be tested.
| Supernatant | 100 | 100 | 100 100 |
| Color Developing Solution | 100 100 | 100 | 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 |
Vortex and mix well, let it stand at room temperature for 10 min, and then measure the absorbance value A at 550 nm with a glass cuvette/96-well plate, which is recorded as A blank, A measurement and A standard respectively, and calculate ΔA standard = A standard - A blank, ΔA measurement = A measurement - A blank. The standard curve and blank tube should be measured only 1~2 times.
Calculation of NO content
1、Drawing of standard curve:
Take the concentration of each standard solution as X-axis (X, μmol/mL), and its corresponding ΔA standard as Y-axis (Y, ΔA standard), and plot the standard curve to get the standard equation Y = kX + b, and then bring the ΔA measurement into the equation to get X (μmol/mL).
2、Calculation of NO content:
(1) Calculated according to the protein concentration of the sample
NO content (μmol/mg prot) = X × V sample ÷ (V sample × Cpr) = X ÷ Cpr
(2) Calculated by sample mass
NO content (μmol/g mass) = X × V sample ÷ (W × V sample ÷ V sample total ) = X ÷ W
(3) Calculated by the number of cells in the sample
NO content ( μmol/104 cell) = X × V sample ÷ (V sample × number of cells ÷ total V sample) = X ÷ number of cells
(4) Calculated by sample liquid volume
NO content (μmol/mL) = X × V sample ÷ V sample = X
V Sample: volume of sample added, 0.1 mL; V Sample Total: volume of extraction liquid added, 1 mL; Cpr: sample protein concentration, mg/mL;
W: sample mass, g; cell number: in 104 units, 10,000 cells.
Caveat
1, The kit is stored at 2~8 ℃. Minimum detection limit: 0.0004 μmol/mL, linear range: 0.00078~0.1 μmol/mL. The detection value should fall in the middle of the detection limit to increase the reliability of the data.
2. Try to use fresh samples for testing. Before the experiment, it is recommended to select 2~3 samples with large expected differences for pre-test. If the absorbance value of the sample is not within the measurement range, it is recommended to dilute or increase the sample volume for testing.
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